The Wilms' tumor (wt1) gene encodes a protein referred to as WT1 which is expressed in the nucleus of certain cells and possesses the structural features of a DNA binding transcription factor. As illustrated in FIG. 1 below, the WT1 protein is a 429 amino acid protein [SEQ ID NO:4] which contains four contiguous zinc fingers at the carboxyl-terminus, and a glutamine- and proline-rich region at the amino-terminus. The amino-terminal region of WT1 protein mediates transcriptional suppression or activation in transient transfection assays [Madden et al, Science, 253:1550-1553 (1991); Maheswaran et al, Proc. Natl. Acad. Sci. USA, 90:5100-5104 (1993); S. L. Madden et al, Oncogene, 8:1713-1720 (1993)]. Splice variants of WT1 can produce the protein with a 17 amino acid insert at amino acid 249 and/or a 3 amino acid insert at amino acid 390.
The wt1 gene encoding WT1 protein is located on chromosome 11p13 and has been found to be mutated or deleted in a subset of hereditary and sporadic Wilms' tumors. Recently, high levels of wt1 expression were reported in a variety of tumors such as ovarian carcinomas [Bruenig et al, Cancer Invest., 11:393-399 (1993)], prostate cancer, mesotheliomas [Park et al, cited above], and leukemias [Miwa et al, Leukemia, 6:405-409 (1992), Miyagi et al, Leukemia, 7:970-977 (1992)].
Diagnostic methods for the ovarian carcinomas, mesotheliomas, and leukemias referred to above are based primarily on clinical attributes and histology of tumor specimens. These methods may at times not distinguish between closely related diseases and may lead to inappropriate treatments of patients. For example, in addition to the presence of many histological variants of malignant mesothelioma, there are other lesions that can affect the pleural surface and present a clinical and histological picture quite similar to malignant mesothelioma [R. J. Pisani et al, Mayo Clin. Proc., 63:1234-1244 (1988)]. Additional relatively specific molecular markers that clearly distinguish between clinically similar lesions for malignant mesotheliomas as well as the other cancers would thus be a valuable clinico-pathological tool which will permit a precise diagnosis. This is important since treatment protocols and prognosis for such conditions vary significantly.
Currently available diagnostic tools include rabbit polyclonal antibodies for WT1 protein known in the art. Morris et al, Oncogene, 6:2339-2348 (1991) describe two such antibodies which recognize amino acid fragments spanning amino acids 294-429 of SEQ ID NO:4 and amino acids 85-173 of SEQ ID NO:4, respectively, of the WT1 protein. Another rabbit polyclonal antibody, which recognizes WT1 amino acids 275-429 of SEQ ID NO: 4 was described by Telerman et al, Oncogene, 2545-2548 (1992). Still other WT1 polyclonal antibodies are commercially available, e.g. the rabbit polyclonal antibody SC-192, which is available from Santa Cruz. However, while polyclonal antibodies in general are able to detect WT1 expression, they have disadvantages in their potential for cross-reactivity with closely related proteins which share common domains with the WT1 protein. These polyclonal antibodies by their nature are likely to provide inconsistent results in antigen specificity and binding affinity studies and are not particularly desirable for diagnostic uses.
Additionally, a commercially available mouse monoclonal antibody, DG-10 (Applied BioTechnology) was raised to the zinc finger region of WT1 and is known to cross-react with the Egr1 proteins. Expression of Egr1 proteins is not limited to cells or tissues that express WT1 and is independently regulated from WT1 expression. Therefore, any antibodies raised to the zinc finger domain in the carboxyl terminus of WT1 may not be useful for selective detection of the WT1.
Another anti-WT1 mouse monoclonal antibody has been described by Mundlos et al, Development, 119:1329-1341 (1993). The Mundlos et al antibody is specific for a 17 amino acid sequence insert (See FIG. 1 below), i.e., a splice variant, that is present in only a subpopulation of the alternatively spliced WT1 mRNA messages.
Thus, there exists a need in the art for methods and compositions for detecting and differentially diagnosing conditions characterized by over-expression or inappropriate expression of WT1.